Hepatitis B virus (HBV) is the most prevalent human tumor virus. It is estimated that chronic HBV infection causes 1,000,000 cases of liver cancer annually. HBV must actively replicate within liver cells to maintain its chronic infection. We work to understand how HBV carries out the individual steps by which it replicates its genome in order to understand how this virus contributes to human cancer. To date, most insights into hepadnavirus DNA replication have come through the study of the avian hepadnavirus, duck hepatitis B virus (DHBV). Over the past decade, our laboratory has made multiple contributions to the understanding of hepadnavirus DNA replication by studying DHBV. During this last funding period our most significant progress was made in elucidating the mechanism of the templates switches during the synthesis of plus-strand DNA of DHBV. These analyses have elucidated two fundamental mechanisms that contribute to the template switching during avian hepadnavirus plus-strand synthesis: (1) a local DNA secondary structure that suppresses in situ priming, and therefore contributes to primer translocation; and (2) a long-distance DNA secondary structure that juxtaposes the donor and acceptor templates for primer translocation and circularization. We propose to develop a mechanistic understanding of HBV DNA replication that builds on and exceeds our current understanding of DHBV DNA replication. This project has three aims: (I) To characterize the process of minus-strand synthesis with an emphasis on the mechanism of the template switch after the synthesis of the fourth nucleotide of minus-strand DNA; (II)To characterize the mechanisms that are important for the crucial events during plus-strand DNA synthesis. These events include the specificity of primer generation and the initiation of plus-strand DNA synthesis, the regulation of in situ priming, and the mechanisms of the two plus-strand template exchanges, primer translocation and circularization; and (III)To determine the mechanisms by which cccDNA amplification is regulated.